The everolimus [40-O-(2-hydroxyethyl)-rapamycin] is a novel macrolide immunosuppressant. Everolimus (also known as SDZ-RAD, RAD, Certican®) was developed by Novartis (Nashan B. The role of Certican in the many pathways of chronic rejection. Transplantation Proceedings, 2001, 33: 3215–3230, herein incorporated by reference) in an effort to improve upon rapamycin (Sirolimus), a proliferation signal inhibitor that blocks growth factor-driven transduction signals in the cellular responses to alloantigen (Cottens S, et al. O-Alkylated rapamycin derivatives and their use, particularly as immunosupressants. WO-009409010 28 Apr. 1994, herein incorporated by reference). Everolimus has greater stability and enhanced solubility in organic solvents, as well as more favorable pharmokinetics with fewer side effects than rapamycin (Sirolimus). Everolimus has been used in conjunction with microemulsion cyclosporin (Neoral®, Novartis) to increase the efficacy of the immunosuppressive regime (Kovarik J M, et al. Exposure-response relationship for Certican in de novo kidney transplantation: define a therapeutic range. Transplantation 2002; 73(6): 920–925, herein incorporated by reference).
The complexity of the clinical state, individual differences in sensitivity to immunosuppressive and nephrotoxic effects, has been rather challenging for physicians to balance between therapeutic efficacy and the occurrence of side effects (Wallemacq, Pierre E. Therapeutic monitoring of immunosuppressant drugs. Where are we? Clinical Chemistry and Laboratory Medicine (2004), 42 (11), 1204–1211, herein incorporated by Chemistry and Laboratory Medicine (2004), 42 (11), 1204–1211, herein incorporated by reference). Therapeutic drug monitoring (TDM), defined as the measurement and interpretation of concentration of these drugs in biological fluids, with as a final objective the prediction of organ responses, became an integral part of transplant protocols.
Therapeutic concentration of everolimus was reported (Kovarik et al.) as 3–15 ng/ml, which was consistent with efficacy while minimizing adverse effects in kidney transplantation. Recent data also showed that therapeutic drug monitoring (TDM) of everolimus would benefit heart transplant patients (Starling, Randall C.; et al. Therapeutic drug monitoring for everolimus in heart transplant recipients based on exposure-effect modeling. American Journal of Transplantation (2004), 4(12), 2126–2131, herein incorporated by reference). Everolimus trough levels were stable in the first year post-transplant and averaged 5.2±3.8 and 9.4±6.3 ng/mL in patients treated with 1.5 and 3 mg/day, respectively.
The TDM of everolimus was reported (McMahon L M, et al. High-throughput analysis of Certican (RAD001) and cyclosporin A (CsA) in whole blood by liquid chromatography/mass spectrometry using a semi-automated 96-well solid-phase extraction system. Rapid Comm. Mass Spectrometry 2000; 14: 1965–1971; Brignol N, et al. High-throughput semi-automated 96-well liquid/liquid extraction and liquid chromatography/mass spectrometric analysis of Certican (RAD001) and cyclosporin A (CsA) in whole blood. Rapid Communications in Mass Spectrometry 2002; 15: 1–10; Streit F. et al. Rapid liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of sirolimus, Certican, tacrolimus, and cyclosporin A in whole blood. Clinical Chemistry. 2002; 48(6): 955–958, each of which herein incorporated by reference). However, methods that use HPLC, LC/MS can be impractical for commercial use due to, for example, long sample preparation time, long assay time, high cost, and labor-intensive procedures. For routine TDM of everolimus, the availability of simple automated tests and high throughput clinical analyzers is desirable.